雷射捕陷誘發溶菌酶結晶化機制的螢光顯微光譜研究

Abstract

迄今，我們團隊已經利用雷射捕陷技術成功地控制溶菌酶在過飽和重水溶液中的成核作用以及其結晶的成長。在雷射捕陷的作用下，溶菌酶團簇與分子於聚焦點所產生的高濃度溶菌酶叢集在此過程中扮演了非常重要的角色，溶菌酶的成核作用以及結晶的成長皆發生於這高濃度的叢集中。也就是說，對於這個高濃度溶菌酶叢集形成的動力學和微觀的結構的瞭解是很重要且不可或缺的。在這項研究中，我們於溶菌酶溶液中加入螢光探針，並以螢光量測系統觀測其在雷射捕陷下的動態與機制。 若丹明B作為螢光探針與溶菌酶重水緩衝溶液混合，將一連續波長之近紅外光雷射聚焦於高於溶液的玻璃/溶液界面3微米的位置。此外，另一道400奈米的飛秒雷射作為螢光的激發光源引入到顯微鏡中，並分別聚焦在與近紅外光雷射的焦點橫向距離為10或20微米的位置上。螢光訊號被連結到時間相關單光子計數系統的單光子雪崩檢光二極體所偵測，我們量測此染料摻雜溶液在雷射捕陷狀態下，光子數及螢光衰減的變化。 在近紅外雷射焦點外10和20微米的螢光強度隨著雷射捕陷的時間逐漸增加；相反地，當捕陷光源被關掉後，螢光強度則開始下降。我們合理的認為螢光強度的變化是歸因於大尺寸高濃度溶菌酶叢集的形成與消散。在雷射捕陷的狀態下，螢光生命週期相較於初始溶液來的短；而在關掉捕陷光源後，螢光生命週期逐漸回升且較初始溶液來的長。前者是以因雷射捕陷作用下，可以以於聚焦焦點所產生的溫度梯度分佈的觀點來解釋；而後者則被認為是由於高濃度溶菌酶叢集內結構的變化，這也是結晶往往被限制在焦點周圍附近毫米尺度範圍空間內的原因。此外，我們也依據螢光生命週期實驗所得到的結果來探討所形成的叢集中類液相溶菌酶團簇的微觀結構。我們相信，在探討雷射捕陷作用下的成核作用以及大尺寸叢集形成的學說上，我們的微觀研究創立了一個重要的里程碑。

We have so far demonstrated the control of nucleation and crystal growth of hen egg-white lysozyme (HEWL) by laser trapping at a glass/solution interfacial layer in the supersaturated D2O solution. One of important findings in those studies is that firstly laser trapping forms a large-sized highly-concentrated domain of HEWL liquid-like clusters and then crystal nucleation and crystal growth take place in the dense cluster domain. Namely, it is important and indispensable to understand the formation dynamics and microscopic structure of the dense domain for developing the laser trapping-induced nucleation and crystal growth. In this research, we studied the formation dynamics of the cluster domain by measuring fluorescence of a probe dye molecule added in the HEWL D2O solution under laser trapping conditions. RhB was used as a probe molecule and mixed with a HEWL D2O buffer solution. A continuous-wave near-infrared (NIR) laser beam was focused at a position 3 μm above a glass/solution interface of the solution. A 400-nm femtosecond laser was also introduced to the microscope as a fluorescence excitation light source and focused at a point 10 or 20 μm laterally away from the focal spot of the NIR laser. Fluorescence was detected with a single photon counting avalanche photodiode which was connected to a time-correlated single photon counting system. We measured the photon count and the fluorescence decay under laser trapping conditions. The fluorescence intensity measured at points 10 and 20 μm away from the focus of the NIR laser was gradually increased during the laser trapping. After the trapping laser was switched off, the fluorescence intensity was decreased. We considered that the florescence intensity increase and decrease are ascribed to the formation and dissolution of a large-sized highly-concentrated cluster domain. The fluorescence lifetime showed tendency to become shorter during laser trapping, whereas it was gradually increased after turning off the trapping laser. The former was explained from the viewpoint of temperature distribution generated from the focal spot of the trapping laser. The latter was considered be due to association structure change of the dense cluster domain, which results in the spatial confinement of the crystallization in a millimeter-scale around the focal spot. Furthermore, we discussed the microscopic structure of the liquid-like clusters constituting the domain based on the fluorescence lifetime measurement. We believe that our study will be an important milestone for the microscopic study of laser trapping-induced crystallization and large dense domain formation.